Team:Goettingen/NoteBook w1

From 2013.igem.org

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<span class="date"><img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span>
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<span class="date">07th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png"></span>
<div class="cont">
<div class="cont">
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<p class="timeline-title">Plasmid mini-prep for Part1-7</p>
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<p class="timeline-title" title="Click to fold/extend">Plasmid mini-prep for Part1-7</p>
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<div class="timeline-cont">
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<div class="timeline-cont" style="display: block;">
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<b>Cryo-Stocks of E. coli transformants (C1, parts 1 – 7):</b>
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  <p class="MsoNormal"><b><span lang="EN-US">Cryo-Stocks of <i style="mso-bidi-font-style:
-
<ul>
+
  normal">E. coli </i>transformants (C1, parts 1 – 7):<o:p></o:p></span></b></p>
-
<li>900 μl E.coli ON culture + 100 μl DMSO 100% in special tubes (ask Katrin or Katrin^^)</li>
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  <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:
-
<li>vortex</li>
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  l4 level1 lfo1"><!--[if !supportLists]--><span lang="FR" style="font-family:Symbol;
-
<li>store at – 70 °C (red box)</li>
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  mso-fareast-font-family:Symbol;mso-bidi-font-family:Symbol;mso-ansi-language:
-
</ul>
+
  FR;mso-bidi-font-weight:bold"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
-
<b>Plasmid Mini-Preparation of parts 1 - 7:</b>
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  </span></span></span><!--[endif]--><span lang="FR" style="mso-ansi-language:FR;
-
<p>ON cultures of C1 for parts 1 – 7 with kit “NucleoSpin? Plasmid” von Macherery-Nagel according to manual pp. 16-17 (NucleoSpin? Plasmid protocol for purification of high copy plasmids</p>
+
  mso-bidi-font-weight:bold">900 μl <i style="mso-bidi-font-style:normal">E.coli</i>
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<p>Step 5: recommended washing of silica membrane with buffer AW was performed</p>
+
  ON culture + 100 μl DMSO 100% in special tubes (ask Katrin or Katrin^^)<o:p></o:p></span></p>
-
<p>Step 7: Elution with buffer AE pre-heated to 50 – 60 °C (in future: DON’T elute with this buffer, use pre-heated HPLC-H2O instead! No one knows what’s inside the buffer and its components (EDTA?) could interfere with sequencing and other reactions)</p>
+
  <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:
-
<br / >
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  l4 level1 lfo1"><!--[if !supportLists]--><span lang="EN-US" style="font-family:
-
<p>NanoDrop – Plasmid concentrations</p>
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  Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family:Symbol;mso-bidi-font-weight:
-
<table cellspacing="0">
+
  bold"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
-
<tr><th>Part Number</th><th>c(DNA)[ng/μl]</th><th>A<sub>260</sub>/A<sub>280</sub></th><th>A<sub>260</sub>/A<sub>230</sub></th>
+
  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
-
<tr><td>1</td><td>84.6</td><td>1.94</td><td>2.18</td></tr>
+
  bold">vortex<o:p></o:p></span></p>
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<tr><td>2</td><td>79.8</td><td>1.94</td><td>2.10</td></tr>
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  <p class="MsoNormal" style="margin-left:36.0pt;text-indent:-18.0pt;mso-list:
-
<tr><td>3</td><td>151.0</td><td>1.89</td><td>2.24</td></tr>
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  l4 level1 lfo1"><!--[if !supportLists]--><span lang="EN-US" style="font-family:
-
<tr><td>4</td><td>29.5</td><td>1.91</td><td>2.07</td></tr>
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  Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family:Symbol;mso-bidi-font-weight:
-
<tr><td>5</td><td>154.9</td><td>1.88</td><td>2.25</td></tr>
+
  bold"><span style="mso-list:Ignore">·<span style="font:7.0pt &quot;Times New Roman&quot;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
-
<tr><td>6</td><td>88.9</td><td>1.92</td><td>2.08</td></tr>
+
  </span></span></span><!--[endif]--><span lang="EN-US" style="mso-bidi-font-weight:
-
<tr><td>7</td><td>5.9</td><td>14.08</td><td>1.87</td></tr>
+
  bold">store at – 70 °C (red box)<o:p></o:p></span></p>
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</table>
+
  <p class="MsoNormal"><b><span lang="EN-US">Backup plates:<o:p></o:p></span></b></p>
-
<p>Stored in red box at - 20°C</p>
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  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">Storage
-
<b>Primers arrived!</b>
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  at 4 °C<o:p></o:p></span></p>
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<p>iGEM_32 ~ 37: dissolved in HPLC water, stored at -20oC in our box</p>
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  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold"><o:p>&nbsp;</o:p></span></p>
-
<p>100uM stock , for PCR dilute 1:20 in HPLC water.</p>
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  <p class="MsoNormal"><b><span lang="EN-US">Plasmid Mini-Preparation of parts 1 -
-
<p>Primer 32 and 33 are strong in forming 2nd structures, increase the amount in PCR.</p>
+
  7:<o:p></o:p></span></b></p>
 +
  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">ON
 +
  cultures of C1 for parts 1 – 7 with kit “NucleoSpin? Plasmid” von
 +
  Macherery-Nagel according to manual pp. 16-17 (NucleoSpin? Plasmid protocol
 +
  for purification of high copy plasmids)<o:p></o:p></span></p>
 +
  <p class="MsoNormal"><u><span lang="EN-US" style="mso-bidi-font-weight:bold">Step
 +
  5:</span></u><span lang="EN-US" style="mso-bidi-font-weight:bold"> recommended
 +
  washing of silica membrane with buffer AW was performed<o:p></o:p></span></p>
 +
  <p class="MsoNormal"><u><span lang="EN-US" style="mso-bidi-font-weight:bold">Step
 +
  7:</span></u><span lang="EN-US" style="mso-bidi-font-weight:bold"> <b><u><span style="color:#92D050">Elution</span></u></b> with buffer AE pre-heated to 50
 +
  – 60 °C <span style="color:red">(in future: DON’T elute with this buffer, </span><b><u><span style="color:#92D050">use pre-heated HPLC-H<sub>2</sub>O</span></u></b><span style="color:red"> instead! No one knows what’s inside the buffer and its
 +
  components (EDTA?) could interfere with sequencing and other reactions)<o:p></o:p></span></span></p>
 +
  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold"><o:p>&nbsp;</o:p></span></p>
 +
  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">NanoDrop
 +
  – Plasmid concentrations<o:p></o:p></span></p>
 +
  <table class="MsoNormalTable" border="1" cellspacing="0" cellpadding="0" style="border-collapse:collapse;border:none;mso-border-alt:solid windowtext .5pt;
 +
  mso-padding-alt:0cm 5.4pt 0cm 5.4pt;mso-border-insideh:.5pt solid windowtext;
 +
  mso-border-insidev:.5pt solid windowtext">
 +
  <tbody><tr style="mso-yfti-irow:0;mso-yfti-firstrow:yes">
 +
    <td width="100" valign="top" style="width:74.7pt;border:solid windowtext 1.0pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><b><span lang="EN-US">Part no.<o:p></o:p></span></b></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border:solid windowtext 1.0pt;
 +
    border-left:none;mso-border-left-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><b><span lang="EN-US">c(DNA) </span></b><b><span lang="EN-US" style="font-family:Calibri">[</span><span lang="EN-US">ng/μl]<o:p></o:p></span></b></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border:solid windowtext 1.0pt;
 +
    border-left:none;mso-border-left-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><b><span lang="EN-US">A<sub>260</sub>/A<sub>280</sub><o:p></o:p></span></b></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border:solid windowtext 1.0pt;
 +
    border-left:none;mso-border-left-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><b><span lang="EN-US">A<sub>260</sub>/A<sub>230</sub><o:p></o:p></span></b></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:1">
 +
    <td width="100" valign="top" style="width:74.7pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">1<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">84.6<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">1.94<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">2.18<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:2">
 +
    <td width="100" valign="top" style="width:74.7pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">2<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">79.8<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">1.94<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">2.10<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:3">
 +
    <td width="100" valign="top" style="width:74.7pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">3<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">151.0<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">1.89<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">2.24<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:4">
 +
    <td width="100" valign="top" style="width:74.7pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">4<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">29.5<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">1.91<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">2.07<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:5">
 +
    <td width="100" valign="top" style="width:74.7pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">5<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">154.9<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">1.88<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">2.25<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:6">
 +
    <td width="100" valign="top" style="width:74.7pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">6<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">88.9<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">1.92<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">2.08<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:7;mso-yfti-lastrow:yes">
 +
    <td width="100" valign="top" style="width:74.7pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">7<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">5.9<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">14.08<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="100" valign="top" style="width:74.75pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal" align="center" style="text-align:center"><span lang="EN-US" style="mso-bidi-font-weight:bold">1.87<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  </tbody></table>
 +
 
 +
  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">Stored
 +
  in red box at - 20°C<o:p></o:p></span></p>
 +
  <p class="MsoNormal"><b><span lang="EN-US">Primers arrived!<o:p></o:p></span></b></p>
 +
  <p class="MsoNormal"><span lang="EN-US">iGEM_32 ~ 37: dissolved in HPLC water,
 +
  stored at -20<sup>o</sup>C in our box<o:p></o:p></span></p>
 +
  <p class="MsoNormal"><span lang="EN-US">100uM stock , for PCR dilute 1:20 in HPLC
 +
  water.<o:p></o:p></span></p>
 +
  <p class="MsoNormal"><i><span lang="EN-US">Primer 32 and 33 are strong in forming
 +
  2<sup>nd</sup> structures, increase the amount in PCR<o:p></o:p></span></i></p>
 +
 
 +
 
 +
 
</div>
</div>
</div>
</div>
</div>
</div>
-
 
-
 
<div class="tlob" id="tl_0606">
<div class="tlob" id="tl_0606">
-
<span class="date">06th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png" /></span>
+
<span class="date">06th<img src="https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png"></span>
<div class="cont">
<div class="cont">
-
<p class="timeline-title">Pick the colonies of part1-7</p>
+
<p class="timeline-title" title="Click to fold/extend">Pick the colonies of part1-7</p>
-
<div class="timeline-cont">
+
<div class="timeline-cont" style="display: block;">
-
<b>Media preparation</b>
+
-
<p>1000ml LB+Amp Solid medium => about 50 Plates(with black code)</p>
+
  <p class="MsoNormal"><b><span lang="EN-US">Media preparation:<o:p></o:p></span></b></p>
-
<b>Transformation</b>
+
  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">1000ml
-
<table cellspacing="0">
+
  LB+Ampicillin Agar =&gt; ca. 50 Plates (Black Code)<o:p></o:p></span></p>
-
<tr><th>Entry Numer</th><th>Location on the kit</th></tr>
+
  <p class="MsoNormal"><b><span lang="EN-US">Transformation:<o:p></o:p></span></b></p>
-
<tr><td>BBa_B0034</td><td>P5 2 M</td></tr>
+
  <table class="MsoNormalTable" border="1" cellspacing="0" cellpadding="0" style="border-collapse:collapse;border:none;mso-border-alt:solid windowtext .5pt;
-
</table>
+
  mso-padding-alt:0cm 3.5pt 0cm 3.5pt;mso-border-insideh:.5pt solid windowtext;
-
<b>Pick the colonies of Part1-7</b>
+
  mso-border-insidev:.5pt solid windowtext">
-
<p>4ml LB with antibiotics, overnight culture for mini-prep[marked with C1]</p>
+
  <tbody><tr style="mso-yfti-irow:0;mso-yfti-firstrow:yes">
-
<p>Backup plates:marked with C1,C2,C3 for each part.</p>
+
    <td width="199" valign="top" style="width:149.45pt;border:solid windowtext 1.0pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">Number<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="199" valign="top" style="width:149.5pt;border:solid windowtext 1.0pt;
 +
    border-left:none;mso-border-left-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">whereabout<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:1;mso-yfti-lastrow:yes">
 +
    <td width="199" valign="top" style="width:149.45pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">B0034<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="199" valign="top" style="width:149.5pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">P5 2
 +
    M<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  </tbody></table>
 +
 
 +
  <p class="MsoNormal"><b><span lang="EN-US">Colonies on the plate: parts 1- 7:<o:p></o:p></span></b></p>
 +
  <p class="MsoNormal"><span lang="EN-US">4ml LB with antibiotics, overnight
 +
  culture for mini-prep[C1]<o:p></o:p></span></p>
 +
  <p class="MsoNormal"><span lang="EN-US">Backup plates: C1,C2,C3 for each part<span style="mso-bidi-font-weight:bold"><o:p></o:p></span></span></p>
 +
 
 +
 
</div>
</div>
</div>
</div>
</div>
</div>
 +
<div class="tlob" id="tl_0605">
<div class="tlob" id="tl_0605">
Line 64: Line 322:
<p class="timeline-title">Preparation of the medium, antibioticks, Transformation.</p>
<p class="timeline-title">Preparation of the medium, antibioticks, Transformation.</p>
<div class="timeline-cont">
<div class="timeline-cont">
-
<b>Preparation of Antibiotic Stocks</b>
+
 
-
<ul>
+
  <p class="MsoNormal"><b><span lang="EN-US">Preparation of Antibiotic Stocks<o:p></o:p></span></b></p>
-
<li>1000x Ampicillin 10 1mL Stocks (EPs in the red box in -20 freezer)</li>
+
  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">1000x
-
<li>1000x Chloramphenicol 10mL Stock (Falcon in Freezer)</li>
+
  Ampicillin 10 1mL Stocks (Freezer red box)<o:p></o:p></span></p>
-
</ul>
+
  <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">1000x
-
<b>Media preparation</b>
+
  Chloramphenicol 10mL Stock (Falcon in Freezer)<o:p></o:p></span></p>
-
<ul><li>250ml*4 LB media with Cm </li>
+
 
-
<li>250ml*1 LB media with Amp </li>
+
  <p class="MsoNormal"><b><span lang="EN-US">Media preparation:<o:p></o:p></span></b></p>
-
</ul>
+
  <p class="MsoNormal"><b><span lang="EN-US"><span style="mso-spacerun:yes">&nbsp;</span></span></b><span lang="EN-US">250ml *4
-
<b>Primer design</b>
+
  Chl</span></p>
-
<table cellspacing="0">
+
  <p class="MsoNormal"><span lang="EN-US"><span style="mso-spacerun:yes">&nbsp;</span><i>[<span class="GramE">use</span> the
-
<tr><th>Code Name</th><th>   Description</th></tr>
+
  ones marked with “5<sup>th.</sup>.6.13 LB<sup>chl</sup>” on EVERY
-
<tr><td>iGEM_36</td><td>   DarR operator sequence + biobrick prefix</td></tr>
+
  plate first. ]*</i></span></p>
-
<tr><td>iGEM_37</td><td>   DarR operator sequence + biobrick sufix</td></tr>
+
  <p class="MsoNormal"><span lang="EN-US"><span style="mso-spacerun:yes">&nbsp;</span>250ml *1 Amp<span style="mso-spacerun:yes">&nbsp; </span>(marked with black) </span></p>
-
</table><br />
+
 
-
<b>Transformation</b>
+
  <p class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span lang="EN-US">primer
-
<table cellspacing="0">
+
  design</span></b><span lang="EN-US">&lt;ordered&gt;</span></p>
-
<tr><th>Biobrick entry number</th><th>Mark</th></tr>
+
  <table class="MsoNormalTable" border="1" cellspacing="0" cellpadding="0" style="border-collapse:collapse;mso-table-layout-alt:fixed;border:none;
-
<tr><td>BBa_J23117</td><td>   part 1</td></tr>
+
  mso-border-alt:solid windowtext .5pt;mso-padding-alt:0cm 3.5pt 0cm 3.5pt;
-
<tr><td>BBa_J23116</td><td>   part 2</td></tr>
+
  mso-border-insideh:.5pt solid windowtext;mso-border-insidev:.5pt solid windowtext">
-
<tr><td>BBa_J23110</td><td>   part 3</td></tr>
+
  <tbody><tr style="mso-yfti-irow:0;mso-yfti-firstrow:yes;height:15.0pt">
-
<tr><td>BBa_J23118</td><td>   part 4</td></tr>
+
    <td width="104" valign="bottom" style="width:78.0pt;border:solid windowtext 1.0pt;
-
<tr><td>BBa_J61101</td><td>   part 5</td></tr>
+
    mso-border-alt:solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt;
-
<tr><td>BBa_BE0240-Cm</td><td>   part 6 -- Cm</td></tr>
+
    height:15.0pt">
-
<tr><td>BBa_B0015</td><td>   part 7 -- Cm</td></tr><tr>
+
    <p class="MsoNormal" align="left" style="text-align:left;mso-pagination:widow-orphan"><span lang="DE" style="font-size:11.0pt;font-family:Calibri;mso-fareast-font-family:
-
<td>BBa_B0034</td><td>   part 8</td></tr>
+
    &quot;Times New Roman&quot;;color:black;mso-font-kerning:0pt;mso-ansi-language:DE;
-
</table>
+
    mso-fareast-language:DE">iGEM_36<o:p></o:p></span></p>
-
Resuspended but not transformed
+
    </td>
-
<table>
+
    <td width="522" valign="bottom" style="width:391.6pt;border:solid windowtext 1.0pt;
-
<tr><th>Entry number</th><th>   location on kit</th></tr>
+
    border-left:none;mso-border-left-alt:solid windowtext .5pt;mso-border-alt:
-
<tr><td>BBa_E0204-Amp</td><td>   P5 12 M</td></tr>
+
    solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt;height:15.0pt">
-
<tr><td>BBa_QO3121</td><td>   P5 20 N</td></tr>
+
    <p class="MsoNormal"><span lang="EN-US" style="font-size:10.0pt;font-family:
-
</table>
+
    &quot;Courier New&quot;;color:black">DarR operator sequence + prefix<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:1;mso-yfti-lastrow:yes;height:15.0pt">
 +
    <td width="104" valign="bottom" style="width:78.0pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt;height:15.0pt">
 +
    <p class="MsoNormal" align="left" style="text-align:left;mso-pagination:widow-orphan"><span lang="DE" style="font-size:11.0pt;font-family:Calibri;mso-fareast-font-family:
 +
    &quot;Times New Roman&quot;;color:black;mso-font-kerning:0pt;mso-ansi-language:DE;
 +
    mso-fareast-language:DE">iGEM_37<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="522" valign="bottom" style="width:391.6pt;border-top:none;
 +
    border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt;
 +
    height:15.0pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:10.0pt;font-family:
 +
    &quot;Courier New&quot;;color:black">DarR operator sequence + suffix<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  </tbody></table>
 +
  <p class="MsoNormal"><b><span lang="EN-US"><o:p></o:p></span></b></p>
 +
 
 +
  <p class="MsoNormal"><b><span lang="EN-US">Transformation:<o:p></o:p></span></b></p>
 +
  <table class="MsoNormalTable" border="1" cellspacing="0" cellpadding="0" style="border-collapse:collapse;mso-table-layout-alt:fixed;border:none;
 +
  mso-border-alt:solid windowtext .5pt;mso-padding-alt:0cm 5.4pt 0cm 5.4pt;
 +
  mso-border-insideh:.5pt solid windowtext;mso-border-insidev:.5pt solid windowtext">
 +
  <tbody><tr style="mso-yfti-irow:0;mso-yfti-firstrow:yes">
 +
    <td width="200" valign="top" style="width:149.85pt;border:solid windowtext 1.0pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US">Number</span></p>
 +
    </td>
 +
    <td width="200" valign="top" style="width:149.85pt;border:solid windowtext 1.0pt;
 +
    border-left:none;mso-border-left-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US">Mark</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:1">
 +
    <td width="200" valign="top" style="width:149.85pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US">J23117</span></p>
 +
    </td>
 +
    <td width="200" valign="top" style="width:149.85pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US">1</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:2">
 +
    <td width="200" valign="top" style="width:149.85pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US">J23116</span></p>
 +
    </td>
 +
    <td width="200" valign="top" style="width:149.85pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US">2</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:3">
 +
    <td width="200" valign="top" style="width:149.85pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US">J23110</span></p>
 +
    </td>
 +
    <td width="200" valign="top" style="width:149.85pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US">3</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:4">
 +
    <td width="200" valign="top" style="width:149.85pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US">J23118</span></p>
 +
    </td>
 +
    <td width="200" valign="top" style="width:149.85pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US">4</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:5">
 +
    <td width="200" valign="top" style="width:149.85pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US">J61101</span></p>
 +
    </td>
 +
    <td width="200" valign="top" style="width:149.85pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US">5</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:6">
 +
    <td width="200" valign="top" style="width:149.85pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US">BBa_BE0240-Chl</span></p>
 +
    </td>
 +
    <td width="200" valign="top" style="width:149.85pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US">6 ---Chl</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:7">
 +
    <td width="200" valign="top" style="width:149.85pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US">BBa_B0015</span></p>
 +
    </td>
 +
    <td width="200" valign="top" style="width:149.85pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US">7----Chl</span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:8;mso-yfti-lastrow:yes">
 +
    <td width="200" valign="top" style="width:149.85pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US">B0034</span></p>
 +
    </td>
 +
    <td width="200" valign="top" style="width:149.85pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt">
 +
    <p class="MsoNormal"><span lang="EN-US">8</span></p>
 +
    </td>
 +
  </tr>
 +
  </tbody></table>
 +
  <p class="MsoNormal"><b><span lang="EN-US">Resuspended DNA from iGEM kit </span></b><span lang="EN-US" style="mso-bidi-font-weight:bold">(already stored in red box in
 +
  -20 frigde)<b>:<o:p></o:p></b></span></p>
 +
  <table class="MsoNormalTable" border="1" cellspacing="0" cellpadding="0" style="border-collapse:collapse;mso-table-layout-alt:fixed;border:none;
 +
  mso-border-alt:solid windowtext .5pt;mso-padding-alt:0cm 3.5pt 0cm 3.5pt;
 +
  mso-border-insideh:.5pt solid windowtext;mso-border-insidev:.5pt solid windowtext">
 +
  <tbody><tr style="mso-yfti-irow:0;mso-yfti-firstrow:yes">
 +
    <td width="199" valign="top" style="width:149.45pt;border:solid windowtext 1.0pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">Number<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="199" valign="top" style="width:149.5pt;border:solid windowtext 1.0pt;
 +
    border-left:none;mso-border-left-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">whereabout<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:1">
 +
    <td width="199" valign="top" style="width:149.45pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">BBa_E0204-Amp<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="199" valign="top" style="width:149.5pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">P5 12
 +
    M<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:2;mso-yfti-lastrow:yes">
 +
    <td width="199" valign="top" style="width:149.45pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">BBa_QO3121<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="199" valign="top" style="width:149.5pt;border-top:none;border-left:
 +
    none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="mso-bidi-font-weight:bold">P5 20
 +
    N<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  </tbody></table>
 +
  <p class="MsoNormal"><b><span lang="EN-US"><o:p></o:p></span></b></p>
 +
 
 +
 
</div>
</div>
</div>
</div>
</div>
</div>
 +
<div class="tlob start" id="tl_0604">
<div class="tlob start" id="tl_0604">
Line 106: Line 555:
<p class="timeline-title">Find the correct DNA sequence of DarR and primer design</p>
<p class="timeline-title">Find the correct DNA sequence of DarR and primer design</p>
<div class="timeline-cont">
<div class="timeline-cont">
-
<table cellspacing="0">
+
  <p class="MsoNormal"><b style="mso-bidi-font-weight:normal"><span lang="EN-US">primer
-
<tr><th>Code Name</th><th>  Description</th></tr>
+
  design</span></b><span lang="EN-US">&lt;ordered&gt;, found the correct sequence
-
<tr><td>iGEM_32</td><td>   Forward primer for DarR ORF amplification, with biobrick prefix</td></tr>
+
   for DarR </span></p>
-
<tr><td>iGEM_33</td><td>   Reverse primer for DarR ORF amplification, with biobrick sufix</td></tr>
+
  <table class="MsoNormalTable" border="1" cellspacing="0" cellpadding="0" style="margin-left:2.55pt;border-collapse:collapse;mso-table-layout-alt:
-
<tr><td>iGEM_34</td><td>   Forward primer for DarR ORF PCR amplification sequencing.</td></tr>
+
  fixed;border:none;mso-border-alt:solid windowtext .5pt;mso-padding-alt:0cm 3.5pt 0cm 3.5pt;
-
<tr><td>iGEM_35</td><td>   Reverse primer for DarR ORF PCR amplification sequencing.</td></tr>
+
  mso-border-insideh:.5pt solid windowtext;mso-border-insidev:.5pt solid windowtext">
-
</table>
+
  <tbody><tr style="mso-yfti-irow:0;mso-yfti-firstrow:yes;height:15.0pt">
 +
    <td width="104" valign="top" style="width:78.0pt;border:solid windowtext 1.0pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt;
 +
    height:15.0pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    Calibri;mso-fareast-font-family:&quot;Times New Roman&quot;;color:black;mso-fareast-language:
 +
    DE">iGEM_32<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="427" valign="bottom" style="width:320.0pt;border:solid windowtext 1.0pt;
 +
    border-left:none;mso-border-left-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt;height:15.0pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:10.0pt;font-family:
 +
    &quot;Courier New&quot;;color:black">Primer DarR + Prefix forw.<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:1;height:15.0pt">
 +
    <td width="104" valign="top" style="width:78.0pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt;height:15.0pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    Calibri;mso-fareast-font-family:&quot;Times New Roman&quot;;color:black;mso-fareast-language:
 +
    DE">iGEM_33<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="427" valign="bottom" style="width:320.0pt;border-top:none;
 +
    border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt;
 +
    height:15.0pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:10.0pt;font-family:
 +
    &quot;Courier New&quot;;color:black">Primer DarR + Suffix rev.<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:2;height:15.0pt">
 +
    <td width="104" valign="top" style="width:78.0pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt;height:15.0pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    Calibri;mso-fareast-font-family:&quot;Times New Roman&quot;;color:black;mso-fareast-language:
 +
    DE">iGEM_34<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="427" valign="bottom" style="width:320.0pt;border-top:none;
 +
    border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt;
 +
    height:15.0pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:10.0pt;font-family:
 +
    &quot;Courier New&quot;;color:black">Primer DarR sequencing forw.<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  <tr style="mso-yfti-irow:3;mso-yfti-lastrow:yes;height:15.0pt">
 +
    <td width="104" valign="top" style="width:78.0pt;border:solid windowtext 1.0pt;
 +
    border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:
 +
    solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt;height:15.0pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:
 +
    Calibri;mso-fareast-font-family:&quot;Times New Roman&quot;;color:black;mso-fareast-language:
 +
    DE">iGEM_35<o:p></o:p></span></p>
 +
    </td>
 +
    <td width="427" valign="bottom" style="width:320.0pt;border-top:none;
 +
    border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
    mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
    mso-border-alt:solid windowtext .5pt;padding:0cm 3.5pt 0cm 3.5pt;
 +
    height:15.0pt">
 +
    <p class="MsoNormal"><span lang="EN-US" style="font-size:10.0pt;font-family:
 +
    &quot;Courier New&quot;;color:black">Primer DarR sequencing rev.<o:p></o:p></span></p>
 +
    </td>
 +
  </tr>
 +
  </tbody></table>
 +
  <p class="MsoNormal"><span lang="EN-US"><o:p></o:p></span></p>
</div>
</div>
</div>
</div>
</div>
</div>
 +
 +
</html>
</html>

Revision as of 08:25, 19 August 2013

07th

Plasmid mini-prep for Part1-7

Cryo-Stocks of E. coli transformants (C1, parts 1 – 7):

·         900 μl E.coli ON culture + 100 μl DMSO 100% in special tubes (ask Katrin or Katrin^^)

·         vortex

·         store at – 70 °C (red box)

Backup plates:

Storage at 4 °C

 

Plasmid Mini-Preparation of parts 1 - 7:

ON cultures of C1 for parts 1 – 7 with kit “NucleoSpin? Plasmid” von Macherery-Nagel according to manual pp. 16-17 (NucleoSpin? Plasmid protocol for purification of high copy plasmids)

Step 5: recommended washing of silica membrane with buffer AW was performed

Step 7: Elution with buffer AE pre-heated to 50 – 60 °C (in future: DON’T elute with this buffer, use pre-heated HPLC-H2O instead! No one knows what’s inside the buffer and its components (EDTA?) could interfere with sequencing and other reactions)

 

NanoDrop – Plasmid concentrations

Part no.

c(DNA) [ng/μl]

A260/A280

A260/A230

1

84.6

1.94

2.18

2

79.8

1.94

2.10

3

151.0

1.89

2.24

4

29.5

1.91

2.07

5

154.9

1.88

2.25

6

88.9

1.92

2.08

7

5.9

14.08

1.87

Stored in red box at - 20°C

Primers arrived!

iGEM_32 ~ 37: dissolved in HPLC water, stored at -20oC in our box

100uM stock , for PCR dilute 1:20 in HPLC water.

Primer 32 and 33 are strong in forming 2nd structures, increase the amount in PCR

06th

Pick the colonies of part1-7

Media preparation:

1000ml LB+Ampicillin Agar => ca. 50 Plates (Black Code)

Transformation:

Number

whereabout

B0034

P5 2 M

Colonies on the plate: parts 1- 7:

4ml LB with antibiotics, overnight culture for mini-prep[C1]

Backup plates: C1,C2,C3 for each part

05th

Preparation of the medium, antibioticks, Transformation.

Preparation of Antibiotic Stocks

1000x Ampicillin 10 1mL Stocks (Freezer red box)

1000x Chloramphenicol 10mL Stock (Falcon in Freezer)

Media preparation:

 250ml *4 Chl

 [use the ones marked with “5th..6.13 LBchl” on EVERY plate first. ]*

 250ml *1 Amp  (marked with black)

primer design<ordered>

iGEM_36

DarR operator sequence + prefix

iGEM_37

DarR operator sequence + suffix

Transformation:

Number

Mark

J23117

1

J23116

2

J23110

3

J23118

4

J61101

5

BBa_BE0240-Chl

6 ---Chl

BBa_B0015

7----Chl

B0034

8

Resuspended DNA from iGEM kit (already stored in red box in -20 frigde):

Number

whereabout

BBa_E0204-Amp

P5 12 M

BBa_QO3121

P5 20 N

04th

Find the correct DNA sequence of DarR and primer design

primer design<ordered>, found the correct sequence for DarR

iGEM_32

Primer DarR + Prefix forw.

iGEM_33

Primer DarR + Suffix rev.

iGEM_34

Primer DarR sequencing forw.

iGEM_35

Primer DarR sequencing rev.

Retrieved from "http://2013.igem.org/Team:Goettingen/NoteBook_w1"